The long-term goal of this project is to understand the molecular mechanisms governing visual photoreceptor transduction in both vertebrate and invertebrate systems. The effort will be focused on the visual signaling process through the investigation of proteins and their post-translational modification. Key ingredients of the project are the use of state-of-the-art technologies in modern mass spectrometry for the microanalysis of proteins applied to the compound eyes of the fruit fly Drosophila melanogaster. Drosophila offers advanced infrastructure for molecular biological inquiries in many aspects of physiological processes including vision. However, one serious disadvantage of Drosophila as a subject for biochemical inquiries is its small size, making collection of enough material for biochemical analysis tedious. Despite such disadvantage, in vivo dark- and light-adaptation experiments in Drosophila are easier than other larger organisms, which is significant in vision research. More than a decade ago the Principal Investigator applied two-dimensional gel electrophoresis (2-D gel) to the analysis of protein phosphorylation induced by light stimuli in the living eyes of Drosophila. However, direct analysis of proteins from 2-D gels was difficult until recently because of the lack of technologies sensitive enough to analyze proteins at picomolar levels, which corresponds roughly to the amounts of proteins stained on a 2-D gel. A rapid development in mass spectrometry in the last several years has enabled this group to analyze directly the protein spots separated on 2-D gels. This group has been one of the research groups who have vigorously pursued application of mass spectrometry to investigate molecular aspects of cellular signaling. In this continuation proposal, the P.I. plans to further advance our knowledge on the role of protein phosphorylation in visual photoreceptor signaling and to develop application methods of mass spectrometry to ask questions essential for the understanding of cellular signaling processes in vision. The specific aims are: 1) to investigate in vivo the mechanism responsible for the dephosphorylation of phosrestin I, a phosphorylated arrestin homolog of Drosophila, 2) to determine the multiple phosphorylation sites of 80K, an InaD PDZ protein that undergoes reversible phosphorylation, 3) to characterize the protein kinase that is responsible for the phosphosphorylation of 80K in vivo, 4) to obtain evidence to support the hypothesis that multiple phosphorylation of 80K regulates the formation of a signaling complex, and 5) to establish methodology to determine the structure of large peptides and even proteins by in-source decay.